This is a proposal for an F32 award for Dr. Daniel Sirkis, a postdoctoral associate in the Department of Molecular and Cell Biology at the University of California, Berkeley. Dr. Sirkis is a cell biologist focusing on the intracellular trafficking of proteins implicated in neurodegenerative disease. This F32 will provide Dr. Sirkis with the support required to accomplish the following goals: (i) to gain experience in the biochemical reconstitution of vesicle trafficking events; (ii) to learn state-of-the-art genome-editing techniques, such as CRISPR/Cas9-mediated mutagenesis; (iii) to use these techniques to clarify the relationship between the intracellular sorting of the amyloid precursor protein (APP and the generation of its proteolytic cleavage product, amyloid- (A), both of which have been implicated in Alzheimer's disease (AD); and (iv) to gain additional experience in scientific communication and mentoring that will be required for a smooth transition to an independent research career. Dr. Sirkis has chosen to carry out his postdoctoral studies under the sponsorship of Dr. Randy Schekman, a cell biologist with deep expertise in vesicle trafficking and biochemical reconstitution. The proposed research project focuses on the intracellular sorting of APP at the trans-Golgi network (TGN). Because the TGN has been implicated as a major site of A generation, this work has the potential to shed light on pathogenic processes involved in AD. In Aim 1, Dr. Sirkis will determine whether small GTP- binding proteins found to influence A production are involved in APP trafficking at the TGN. In Aim 2, Dr. Sirkis will determine whether the transmembrane protein SorLA, a known risk factor for late-onset AD, influences A production by facilitating APP transport from either the TGN or endosomes. In Aim 3, Dr. Sirkis will determine whether novel AD risk factor phospholipase D3 influences A production via the endoplasmic reticulum or the TGN. The overarching goals of this project are (i) to characterize how novel, Golgi-localized, GTP-binding proteins regulate the transport of APP at the TGN; and (ii) clarify how two genetic risk factors for late-onset AD, both of which are membrane proteins, affect the trafficking of APP. If successful, the proposed studies are expected to increase our understanding of APP sorting and A generation, and by extension, elucidate biological processes underpinning AD. Importantly, Dr. Sirkis' F32 training will prepare him for an independent research career that focuses on the intracellular trafficking of proteins involved in neurodegeneration.